Coupling Ambient Ionization Mass Spectrometry with Liquid Chromatography and Electrochemistry and Their Applications A dissertation presented to the faculty of the College of Arts and Sciences of Ohio University In partial fulfillment of the requirements for the degree Doctor of Philosophy Yi Cai December 2016 © 2016 Yi Cai. All Rights Reserved. 2 This dissertation titled Coupling Ambient Ionization Mass Spectrometry with Liquid Chromatography and Electrochemistry and Their Applications by YI CAI has been approved for the Department of Chemistry and Biochemistry and the College of Arts and Sciences by Hao Chen Associate Professor of Chemistry and Biochemistry Robert Frank Dean, College of Arts and Sciences 3 ABSTRACT CAI, YI, Ph.D., December 2016, Chemistry Coupling Ambient Ionization Mass Spectrometry with Liquid Chromatography and Electrochemistry and Their Applications Director of Dissertation: Hao Chen Ambient ionization methods allow the ionization of untreated samples in the open environment. In this dissertation, two different ambient ionization techniques, desorption electrospray ionization (DESI) and probe electrospray ionization (PESI), has been developed and coupled with liquid chromatograph (LC) and electrochemistry (EC) and their analytical applications have been explored and discussed. Liquid sample DESI generally employs a DESI probe to spray solvent with high voltage to ionize sample as the sample solution is delivered to the ion source by a piece of fused silica transfer capillary. A new splitting interface, a PEEK capillary tube with a micro-orifice drilled in the capillary wall, was used to connect with LC column for applying DESI ionization. A small portion of LC eluent emerging from the orifice can be directly ionized by DESI with negligible time delay while the remaining analytes can be online collected. Furthermore, online derivatization using reactive DESI is possible for additional application such as supercharging proteins. Since splitting via an orifice introduces negligible dead volume and back pressure, the performance of the LC/DESI-MS with the focus of using ultra-fast LC for analyzing sample was further evaluated. Using a monolithic C18 column, metabolites in urine can be separated within 1.6 min, online monitored by DESI and collected as purified samples. 4 Negative ions can be directly generated for acidic analytes in acidic LC eluent by DESI during the LC/MS analysis process using a spray solvent with alkaline pH. In addition, DESI-MS is found to be compatible with ultra-performance liquid chromatography (UPLC) for the first time. The 45 s separation of drugs can be achieved via UPLC/DESI- MS under high temperature. The combination with EC further broadens LC/MS applications. UPLC-MS combined with EC via DESI was first developed for the structural analysis of proteins/peptides that contain disulfide bonds. Using this combined UPLC/EC/DESI-MS method, peptides containing disulfide bonds can be differentiated from those without disulfide bonds, as the former are electroactive and reducible. MS/MS analysis of disulfide-reduced peptide ions provides increased information about the peptide sequence and disulfide-linkage pattern. In addition, upon online electrolytic reduction and reactive DESI, supercharged proteins showed increased charges distribution which is of value for MS/MS sequencing application. PESI, another ambient ionization technique, employs a conductive solid probe to ionize samples directly on the probe tip with the aid of applied high voltage. Due to the high salt tolerance of PESI, the detection of electrochemical reaction products in room- temperature ionic liquids is realized, for the first time. Furthermore, PESI-MS allows the detection the electrochemical reaction products on different or multiple electrode surfaces. In addition, peptides and proteins fractionated through isoelectric focusing (IEF) can also be directly analyzed by PESI-MS. 5 DEDICATION I dedicate this dissertation to my friends, parents, and husband 6 ACKNOWLEDGMENTS I acknowledge my advisor, Dr. Hao Chen, for his great mentoring. His dedication to science deeply impacts me and encourages me to become a good researcher. He is also generous to support me for attending conferences. I acknowledge to all my dissertation committee members, Dr. Peter de B. Harrington, Dr. Shiyong Wu and Dr. Shigeru Okada for their guidance. I acknowledge to National Science Foundation Career Award (to Dr. Hao Chen, CHE-1149367), National Science Foundation Instrument Development for Biological Research (CHE 1455554), National Natural Science Foundation of China (Grant 21328502), Edison Biotechnology Institute Faculty Fellowship, Merck Research Laboratories New Technology Review & Licensing Committee, Ohio Third Frontier Technology Validation and Start-Up Fund, and Center for Intelligent Chemical Instrumentation, Department of Chemistry and Biochemistry, Ohio University for the financial support. I acknowledge to all former and current members in Dr. Hao Chen’s group, Dr. Zhixin Miao, Dr. Yun Zhang, Zongqian Yuan, Pengyuan Liu, Mei Lu, Dr. Qiuling Zheng, Si Cheng, He Xiao, Meihong Hu, Chang Xu, Yuexiang Zhang, Najah Almowalad, Amanda Forni, Sabrina Cramer, Fengyao Li, Denial Adams, David Hu, Prof. Dr. Ping Li, Dr. Ning Pan, Prof. Dr. Kehua Xu, Prof. Dr. Jun Wang, Prof. Dr. Qiuhua Wu and Prof. Dr. Zhi Li for their assistance and support. I acknowledge to all my collaborators, Dr. Howard D. Dewald (Ohio University); Dr. Michael Held (Ohio University); Dr. Huifang Yao (Merck & Co., Inc., Rahway, 7 New Jersey), Dr. Yong Liu (Merck & Co., Inc., Rahway, New Jersey), Dr. Roy Helmy (Merck & Co., Inc., Rahway, New Jersey) for their helpful discussion and suggestions. I acknowledge to Dr. Andrew Tangonan, Bascom French, Paul Schmittauer, Aaron Dillon, Carolyn Khurshid, Marlene Jenkins, Jackie Bennett-Hanning, Dr. Zhengfang Wang, Dr. Mengliang Zhang, Xue Zhao, Xinyi Wang, and Dr. Lei Wang for their generous help. 8 TABLE OF CONTENTS Page Abstract ............................................................................................................................... 3 Dedication ........................................................................................................................... 5 Acknowledgments............................................................................................................... 6 List of Figures ................................................................................................................... 11 List of Schemes ................................................................................................................. 16 List of Abbreviations ........................................................................................................ 17 Chapter 1: Introduction ..................................................................................................... 19 1.1 Mass Spectrometry ................................................................................................. 19 1.2 Mass Spectrometer .................................................................................................. 19 1.3 Ionization Methods ................................................................................................. 20 1.3.1 ESI-MS ............................................................................................................ 21 1.3.2 DESI-MS .......................................................................................................... 22 1.3.3 Liquid Sample DESI-MS ................................................................................. 24 1.3.4 Probe Electrospray Ionization (PESI) .............................................................. 25 1.4 EC-MS .................................................................................................................... 26 1.4.1 EC/ESI-MS ...................................................................................................... 28 1.4.2 EC/DESI-MS ................................................................................................... 28 1.5 LC/DESI-MS .......................................................................................................... 30 Chapter 2: A New Splitting Method for Both Analytical and Preparative LC/MS .......... 33 2.1 Introduction ............................................................................................................. 33 2.2 Experimental ........................................................................................................... 35 2.2.1 Chemicals ......................................................................................................... 35 2.2.2 LC Separation Condition ................................................................................. 36 2.2.3 DESI-MS Detection ......................................................................................... 36 2.3 Results and Discussion ........................................................................................... 37 2.3.1 Protein Detection ............................................................................................. 37 2.3.2 Saccharide Detection........................................................................................ 46 2.3.3 Sulfonamides Detection ................................................................................... 49 2.4 Conclusions ............................................................................................................